Method and apparatus for nucleic acid hybridization

ABSTRACT

A method and apparatus designed for nucleic acid hybridization employs a hydrogen bond denaturation area with a higher temperature and a lower temperature nucleic acid hybridization area that is immobilized with nucleic acid probes. Nucleic acid-containing samples are introduced into the hydrogen bond denaturation area for heating curled nucleic acids in samples so that they become linear and are guided into the nucleic acid hybridization area. In this area, the nucleic acid hybridization rate can be multiplied by increasing the kinetic energy by the repeated flow of driven fluid. The nucleic acid hybridization apparatus provided by the invention contains a hydrogen bond denaturation area, a nucleic acid hybridization area, a two-way driving apparatus, and a temperature control element. The temperatures in the hydrogen bond denaturation area and the nucleic acid hybridization area can be maintained through the management of this temperature control element, and the fluid can gain needed kinetic energy through the two-way driving pump.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention generally relates to a method for nucleic acidhybridization, and specifically relates to a method and apparatus withtemperature control for nucleic acid hybridization and hydrogen bonddenaturation to accelerate nucleic acid hybridization.

[0003] 1. Related Art

[0004] Based on rapidly developed techniques, such as PCR (polymerasechain reaction) and nucleic acid hybridization, molecular biotechnologyhas been gradually integrated with different areas, including materialscience, bioinformatics, and electronic technology, to bring about a newarea of Biochips. The emergence of bio chips could significantlydecrease needed detection time for some diseases. For example, theinitial detection time of 3-5 days by cell culture techniques can bedecreased to be less than 6 hours by utilizing biochip technology.However, such 6-hour detection time still cannot satisfy the demands ofcertain diseases. For instance, some diseases take less than 2 days fromdiagnosis to cause death. Therefore, current bio chip research has beenfocused on shortening the needed detection time of the biochip.

[0005] To shorten the detection time, it is necessary to start from themost time-consuming technique during the test. PCR takes approximately1.5 hours and nucleic acid hybridization takes about 4 hours Together,these two techniques account for 92% of the whole biochip detectiontime. Therefore, how to shorten the amount of time consumed by these twotechniques has become a key point. Presently, numerous researchers haveput forth much effort on this issue, and the invention is focused on themost time-consuming nucleic acid hybridization technique.

[0006] The underlying mechanism for techniques such as HybridizationHelper is to utilize an oligonucleoride (i.e. Helper), which iscomplementary to the upstream or downstream region of the area whereprobing nucleic acid is hybridized to sample nucleic acid. Suchhybridization between the nucleic acid Helper and sample nucleic acid isemployed to stretch the sample nucleic acid (to eliminate the originalcluttered circular conformation that is unfavorable for hybridization),which help the hybridization reaction.

[0007] In addition, another technique (called nucleic acid precipitatingreagents) is available. The underlying mechanism for this technique isto utilize various buffered salt solutions to promote the sample nucleicacid precipitation at the nearby area of the probing nucleic acid. Suchmethodology increases the sample nucleic acid concentration in the localarea to promote the processing of the hybridization reaction.

[0008] There is still another available technique called the branchedoligonucleoride multimer technique, which uses probes immobilized on thesurface of a chip for catching sample nucleic acid. Then, a branchedoligonucleoride multimer, which is complementary to the sample nucleicacid, is linked to sample nucleic acid. Finally, fluor- or radio-labelednucleic acid detectors are complementarily hybridized with the branchedoligonucleoride multimer. Since tens or hundreds of detectors canhybridize onto a branched oligonucleoride multimer, the detectionintensity can be greatly increased and the hybridization time cantherefore be

[0009] Furthermore, a technique called electrically controlledhybridization takes advantage of the characteristics of negativelycharged nucleic acid. By immobilizing the positive pole near the nucleicacid probe, nucleic acid in samples is lured near to the probe to makethe sample nucleic acid highly concentrated in a small area andaccomplish the goal of accelerating hybridization.

[0010] Another technique called volume exclusion agents employs organicmolecules to form a reticular macro-structure, which can expel part ofthe hybridization buffer to increase the local concentration of samplenucleic acid and therefore promote the hybridization reaction.

[0011] Similar to the above technique, one technique called amphipathichydrocarbon polymer (AHP) utilizes bipolar organic molecules(hydrophilic and hydrophobic) to form a reticular macro-structure. Thismacrostructure can expel part of the hybridization buffer to increasethe local concentration of sample nucleic acid and therefore promote thehybridization reaction.

[0012] An apparatus called the highly parallel-integrated microfluidicbiochannel array has integrated various functions, including samplepretreatment, PCR, hybridization, washing, and signal detection.However, the hybridization rate of this apparatus has not yet beenimproved.

[0013] Finally, in the apparatus called the dynamic hybridizationsystem, a nucleic acid probe is fixed on a semipermeable membrane.Meanwhile, fluid (containing sample nucleic acid) is driven by air orvacuum compression to flow towards the semipermeable membrane. Samplenucleic acids are delayed when the fluid passes through thesemipermeable membrane and sample nucleic acids accumulate around themembrane to yield a higher concentration of sample nucleic acid. Thehybridization rate is increased because non-complementary nucleic acidis able to pass through the holes of the semipermeable membrane.

[0014] Most of the above mentioned methods increase the hybridizationrate by increasing sample nucleic acid concentration, linearlizingsamples, or employing branched structure. All these hybridizationaccelerating methods have limitations, such as only being operable on alarge scale. Nevertheless, how to speed up nucleic acid hybridizationwhile also making the process applicable on a small scale is still thefocus of a great deal of effort in research.

SUMMARY OF THE INVENTION

[0015] In order to solve the problems of the above-mentioned knowntechniques, this invention provides a method and apparatus designedspecifically for nucleic acid hybridization, which fulfills the aim ofaccelerating the nucleic acid hybridization rate by increasing kineticenergy and thermal energy of the nucleic acid-containing flow.

[0016] Introducing a novel nucleic acid hybridization method is theother aim of this invention. By increasing the thermal energy of nucleicacid-containing fluid, originally curled nucleic acid is linearlized andthe hybridization rate is increased.

[0017] To accomplish these aims, the invention provides a method fornucleic acid hybridization. The method includes the following steps.Firstly, providing a nucleic acid hybridization area of the firsttemperature, which is the first channel immobilized with several nucleicacid probes. Secondly, providing a hydrogen bond denaturation area ofthe second temperature, which is the second channel attached with anucleic acid hybridization area to form a connecting channel. Thirdly,guiding the nucleic acids-containing fluid into the connection channel.Finally, driving such fluid to repeatedly pass through the first channeland the second channel.

[0018] A two-way driving apparatus, such as a two-way air-driven orfluid-driven pump, is used to drive the fluid. The fluid is retained inthe first channel for the first period (such as 3 minutes) and thendriven to the second channel for the second period (such as 10 seconds).While being retained in the first channel, the kinetic energy of thefluid is increased by being driven to flow back and forth.

[0019] The invention further provides a nucleic acid hybridizationapparatus, including a hydrogen bond denaturation area equipped with afirst channel for denaturing hydrogen bonds of nucleic acids; and anucleic acid hybridization area equipped with a second channel, which isimmobilized with nucleic acid probes. A connecting channel is formedbetween the nucleic acid hybridization area and the first channel. Atemperature control element is used for maintaining the temperature ofthe hydrogen bond denaturation area and nucleic acid hybridization areaat the first and second temperatures, respectively. A two-way drivingelement is used for driving the flow of the nucleic acids-containingfluid that is infused in the connecting channel.

[0020] Nucleic acid probes for hybridization can be DNA, RNA, peptide,peptide-RNA complex or derivatives of peptide-RNA complex. Thetemperature control element can then keep a stable temperature for thehydrogen bond denaturation area 10 and the nucleic acid hybridizationarea 20. The energy source can be an electric heater, microwave, laseror light.

[0021] To achieve the above-mentioned and other objects, features andadvantages of the invention clearer and easier to understand, severalpractical examples are described in detail below, together with attachedfigures:

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram of the nucleicacid hybridization method of the invention; and FIG. 2 is a diagram ofthe nucleic acid hybridization apparatus of the invention. DETAILEDDESCRIPTION OF THE INVENTION

[0022] First of all, the operation principal of the invention is toincrease the hybridization rate by elevating the kinetic energy andthermal energy of the nucleic acid-containing fluid. To be morespecific, making the nucleic acid-containing sample flow back and forthincreases the nucleic acid extraction rate. Besides, together with anincrease in thermal energy to break the intramolecular hydrogen bond ofnucleic acid molecules, originally curled nucleic acid molecules becomelinear and therefore favorable for hybridization.

[0023] Based on the foregoing principles, the invention discloses amethod with two different nucleic acid reaction areas. The first area isa nucleic acid hybridization area and the second area is a hydrogen bonddenaturation area. The temperature of the hydrogen bond denaturationarea is higher to denature the intramolecular hydrogen bond of nucleicacids. The temperature of the nucleic acid hybridization area is lower,and in this area the heated linear nucleic acid can cross-hybridize withcomplementary nucleic acid probes to accelerate hybridization rates.Besides, an element is used to drive fluid back and forth and increasethe kinetic energy of the fluid. The kinetic energy of the nucleic acidin the nucleic acid hybridization area can then be increased bycontinuing back and forth flow. Therefore, hybridized nucleic acid cangain kinetic energy and thermal energy to accelerate the hybridizationrate, which is the purpose of the invention.

[0024] Consequently, the nucleic acid hybridization method of theinvention includes cycles of two stages. The first one is for increasingthe thermal energy of nucleic acid molecules and denaturingintermolecular hydrogen bonds of nucleic acids. After completing thisstage, the next stage, i.e. the nucleic acid hybridization stage, isprocessed. In the nucleic acid hybridization stage, through the rapidback and forth flow of samples (containing nucleic acid with a denaturedhydrogen bond) to rapidly react with immobilized nucleic acid probes inthis area, rapid hybridization can be accomplished. For this reason, theperiod for samples to stay in the hydrogen bond denaturation area can beonly long enough for complete denaturation of the hydrogen bond.Adequate time is allotted for samples to stay in the nucleic acidhybridization area to allow existing nucleic acid molecules with adenaturing hydrogen bond to properly react. After arriving at thenucleic acid hybridization area of lower temperature, nucleic acidmolecules with denatured hydrogen bonds are gradually restored back totheir original status. Therefore, these two stages have to be processedrepeatedly until the desired level of nucleic acid hybridization hasbeen achieved.

[0025] Numerous nucleic acid probes are easily immobilized due to theirmolecular structure, which is able to bind to glass substrates.Immobilized nucleic acid probes can rapidly react with samplescontaining target nucleic acid (with denatured hydrogen bonds) flowingback and forth. The back and forth flowing rate of the sample can beeasily controlled by the element responsible for the flow of fluid. Thiselement can be driven by a two-way pump, either air-driven orfluid-driven. Practically, two-way air-driven mode is better. The backand forth flow rate in the nucleic acid hybridization area can bebetween 0.1 to 50 rounds per second.

[0026] The period of the first stage, which is for the sample to stay inthe hydrogen bond denaturation area, can be about 10 seconds, anddepends on the inner channel diameter of the hydrogen bond denaturationarea. For the second stage, with samples staying in the nucleic acidhybridization area, the period can be about 3 minutes or shorter. Thetemperature in the hydrogen bond denaturation area can be controlled toaround 80-100° C., at which point intramolecular hydrogen bonds ofnucleic acids can be denatured; 90° C. is optimal. For the nucleic acidhybridization area, the temperature is between 20 to 68° C., with 40° C.being optimal.

[0027] As shown in FIG. 1, the method includes introducing nucleicacid-containing samples from infusion holes 11 or 12 in the hydrogenbond denaturation area, though infusion hole 11 is superior. Throughthis step, samples can be heated to reach a higher temperature to unfoldthe nucleic acid and make it linear. A wiggled channel 12 is located inthe hydrogen bond denaturation area 10. Since this invented methodincreases kinetic and thermal energy in the nucleic acids, the size ofthe channel is not limited. Therefore, the channels can be made in macroor micro scale, as well as unlimited shapes.

[0028] Similarly, the infusion hole 21 and the wiggled channel 22 areavailable in the nucleic acid hybridization area 20. As shown in FIG. 1,the channel 22, located in the nucleic acid hybridization area 20, isinterconnected with the channel 12, located in the hydrogen bonddenaturation area. This interconnection enables the back and forth flowsof samples inside the channel. Likewise, shape or size of the channel 22is not limited. In the nucleic acid hybridization area 20, the surfaceof the channel 22 is immobilized with lots of nucleic acid probes. Whenthose nucleic acids, which are linearlized by thermal energy, increase,flow to the channel surface area where nucleic acid probes areimmobilized, hybridization between nucleic acid and probe occurs.Together with back and forth flow to increase the kinetic energy ofsamples passing probes, the hybridization rate is synergisticallyaccelerated.

[0029] Immobilization of nucleic acid probes on a substrate is by way ofaffinity between the macromolecule base of the probes and the substrate.Basically, the ability to be immobilized with probes is the selectioncriteria for the substrate.

[0030] According to the above-mentioned protocol, the inventiondiscloses an apparatus for nucleic acid hybridization. Please refer toFIG. 2, which includes a micro-pump 40, a valve element 30, a hydrogenbond denaturation area 10, a nucleic acid hybridization area 20 and atemperature control element 50. Among them, the first channel and thesecond channel are located in the hydrogen bond denaturation area andnucleic acid hybridization area, respectively. These two channels areinterconnected to become a connecting channel with the first and secondopen holes, respectively, and are used for sample influx and as entranceand exit in driving the back and forth flow of fluid.

[0031] As shown in FIG. 2, the above-mentioned kinetic energy isprovided by micro-pump 40, including transfer of nucleic acid-containingsamples from the hydrogen bond denaturation area 10 to the nucleic acidhybridization area 20 and the back-and-forth swift flow of samplesinside the channel of the nucleic acid hybridization area 20. The valveelement 30 functions to control the flow direction of the micro-pump 40and then drive the samples to flow from the hydrogen bond denaturationarea 10 to the nucleic acid hybridization area 20, or from the nucleicacid hybridization area 20 to the hydrogen bond denaturation area 10.This valve element 30 can also be integrated into the micro-pump 40.

[0032] A two-way pump can be used as the micro-pump 40. After thesamples inside the hydrogen bond denaturation area have gained enoughthermal energy, and nucleic acids in samples have been linearlized, thesamples are allowed to flow to the nucleic acid hybridization area 20.Meanwhile, the micro-pump 40 is controlled to repeatedly provide tracekinetic energy to enable existing samples to flow back and forth. Suchassistance provided by the micro-pump 40 makes the nucleic acidhybridization of the invention even more efficient.

[0033] In the invention, nucleic acid probes applicable in hybridizationcan be DNA, RNA, peptide, peptide-RNA complex or derivatives ofpeptide-RNA complex. The temperature control element 50 can maintain astable temperature for the hydrogen bond denaturation area 10 and thenucleic acid hybridization area 20. An electric heater, microwave,laser, or light can be used as the heat source.

[0034] The period for hybridization can be shortened from the 4 hours oforiginal techniques to within 30 minutes, or even shorter, by applyingthe nucleic acid hybridization method and apparatus of the invention. Intheory, it can be shortened to be less than 10 minutes.

[0035] The nucleic acid hybridization method and apparatus of theinvention is simply constructed, inexpensive and an initiative elementis not needed.

[0036] The invention being thus described, it will be obvious that thesame may be varied in many ways. Such variations are not to be regardedas a departure from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A nucleic acid hybridization method, comprisingthe steps of: providing a nucleic acid hybridization area of a firsttemperature, which has a first channel immobilized with a plurality ofprobes thereon; providing a hydrogen bond denaturation area of a secondtemperature, which has a second channel interconnected to the firstchannel to form a connection channel; guiding a nucleic acids-containingfluid into the connection channel; driving the fluid to pass the secondchannel and the first channel back and forth.
 2. The nucleic acidhybridization method of claim 1, wherein the first temperature isbetween 20 to 68° C.
 3. The nucleic acid hybridization method of claim1, wherein the optimal temperature of the first temperature is 40° C. 4.The nucleic acid hybridization method of claim 1, wherein the secondtemperature is between 80 to 100° C.
 5. The nucleic acid hybridizationmethod of claim 1 wherein the optimal temperature of the secondtemperature is 90° C.
 6. The nucleic acid hybridization method of claim1, wherein a two-way driving apparatus is utilized for driving thefluid.
 7. The nucleic acid hybridization method of claim 6, wherein thetwo-way driving apparatus is selected from the group of a two-wayair-driven pump and a two-way fluid-driven pump.
 8. The nucleic acidhybridization method of claim 1, wherein a way is selected from thegroup of the hydrogen bond denaturation area and the nucleic acidhybridization area for guiding the fluid into the connection channel,while the optimal way is the hydrogen bond denaturation area.
 9. Thenucleic acid hybridization method of claim 1, wherein the step ofdriving the fluid to pass the second channel and the first channel backand forth is to restrain the fluid in the first channel for a firstperiod and then drive the fluid to the second channel to stay for asecond period.
 10. The nucleic acid hybridization method of claim 9,wherein during the first period for the fluid staying in the firstchannel, the two-way driving apparatus drive the fluid to flow back andforth for increasing the kinetic energy of the fluid.
 11. An nucleicacid hybridization apparatus, comprising: a hydrogen bond denaturationarea having a first channel and a first hole, for denaturing thehydrogen bonds of nucleic acids; a nucleic acid hybridization areahaving a second hole and a second channel immobilized with nucleic acidprobes thereon, the second channel is interconnected with the firstchannel to form a connection channel; a temperature control element, formaintaining hydrogen bond denaturation area and nucleic acidhybridization area at a first and a second temperature respectively; anda two-way driving element, which drives a nucleic acids-containing fluidinfused into the connection channel to flow back and forth through thefirst hole and the second hole.
 12. The nucleic acid hybridizationapparatus of claim 11, wherein the nucleic acid probes is selected fromthe group of DNA, RNA, peptide, peptide-RNA complex and derivatives of13. The nucleic acid hybridization apparatus of claim 11 wherein thetemperature control element is a fixed heat source.
 14. The nucleic acidhybridization apparatus of claim 13 wherein the fixed heat source isselected from the group of an electric heater, a microwave, a laser anda light sources
 15. The nucleic acid hybridization apparatus of claim11, wherein the first temperature is between 20 to 68° C.
 16. Thenucleic acid hybridization apparatus of claim 11, wherein the optimaltemperature of the first temperature is 40° C.
 17. The nucleic acidhybridization apparatus of claim 11, wherein the second temperature isbetween 80 to 100° C.
 18. The nucleic acid hybridization apparatus ofclaim 11, wherein the optimal temperature of the second temperature is90° C.
 19. The nucleic acid hybridization apparatus of claim 19, whereinthe two-way driving element is selected from the group of a two-wayair-driven pump and a two-way fluid-driven
 20. The nucleic acidhybridization apparatus of claim 11, wherein the two-way driving elementdrives the fluid to stay in the first channel for the first period andthen drives the fluid to stay in the second channel for the secondperiod.
 21. The nucleic acid hybridization apparatus of claim 20,wherein during the first period for the fluid staying in the firstchannel, the two-way driving element drives the fluid to flow back andforth for increasing the kinetic energy of the fluid.
 22. The nucleicacid hybridization apparatus of claim 11, wherein further comprising avalve element for controlling back and forth the flowing direction ofthe fluid driven by the two-way driving element.